Abstract There is currently no successful treatment for long-segment tracheal stenosis, most commonly a result of prolonged intubation or tracheostomy. Available treatment options are inadequate in preventing restenosis and often require successive surgeries. A tissue engineered trachea has the potential to fill this need. A functional tracheal replacement must (1) have radial rigidity, (2) be vascularized or encourage vascularization and (3) contain respiratory epithelium to restore an open airway while avoiding restenosis, bacterial infections and ischemic necrosis. This proposal seeks to develop a tracheal replacement with these functional requirements by fusing tissue rings into a composite tissue tube comprised of multiple cell types with controlled, localized growth factor presentation, and to evaluate its ability to serve as a tracheal replacement in a rabbit airway defect model in vivo. The hypothesis of this work is that a multi- cell type, scaffold-free neotrachea can be engineered with vital cellular organization and critical functionality by delivering bioactive factors in a spatiotemporally controlled manner to modular tissue units comprised of self-assembled human cells. By utilizing a custom assembly system, we will integrate the tissue ring units into a composite tubular tracheal construct. Specifically, the proposal aims to (1) engineer cartilaginous ring and tubular structures with defined dimensions and requisite mechanical properties using high-density hMSC culture to serve as radial support in the neotrachea, (2) engineer cartilage-prevascular composite tubular constructs with alternating cartilage and vascular tissue rings to support blood vessel formation in the neotrachea, (3) engineer a luminal epithelial lining on cartilaginous tubes for interfacing with the external environment and (4) test the capacity of the engineered tracheas to restore airway functionality in an animal defect model. This work seeks to engineer a replacement human trachea with radial rigidity that supports vascularization and epithelialization when implanted in vivo. This bottom-up, self- assembled high-cell density strategy with localized bioactive factor presentation is a novel, modular approach to engineer a multi-tissue, function-restoring organ of the respiratory system for patients suffering from large and currently untreatable tracheal defects. This platform technology also has the potential to be employed to regenerate other complex tissues and organs in the body.